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Background T cell receptor (TCR)-based therapies offer a powerful approach to target intracellular tumor antigens that are inaccessible to modalities such as antibody-drug conjugates (ADCs) and conventional antibodies. Central to these therapies are TCRs that can specifically recognize tumor antigen (Ag)-derived peptides presented on MHC molecules. However, identifying TCRs with both high affinity and high specificity remains a significant bottleneck in therapeutic development. Tumor-infiltrating lymphocytes (TILs), which often contain clonally expanded, antigen-experienced T cells, may offer an enriched pool of candidates with ideal characteristics to develop TCR- based T cell engagers.Methods We utilized Repertoire’s proprietary DECODE™ Platform to identify antigen-specific TCRs from TILs and peripheral blood mononuclear cells (PBMCs) derived from melanoma, head and neck, and lung cancer patients. Multiplexed, barcoded peptide-MHC class I tetramer libraries were used to stain TILs and PBMCs, which were then subjected to single-cell sequencing to identify and characterize T cells reactive to tumor associated antigens. The functional avidity of each TCR identified was assessed via cloning and transduction into Jurkat reporter cells with subsequent co-culture with T2 cells across a range of peptide concentrations. TCR specificity was characterized via an alanine scan. Furthermore, soluble TCR-bispecifics were expressed in mammalian cells and purified, and affinity was assessed using biolayer interferometry. Finally, we employed yeast surface display to assess the ability of the TCRs to be used in yeast systems for affinity optimization, characterizing surface expression, and pMHC binding characteristics.Results Our study identified antigen-specific TCRs targeting peptides derived from tumor-associated antigens including gp100, MAGE family members, and other non-canonical, ‘dark’ antigens. Notably, we identified several TCRs specific for the gp100 YLEP peptide presented in HLA-A*02:01. These TCRs, derived from a variety of anatomical locations, donors, and disease contexts, exhibited high similarity in their CDR3 sequences and shared VJ gene usage in both α and β chains, suggesting convergent selection. Tumor-infiltrating T cells exhibited an exhausted phenotype, whereas peripheral T cells displayed an effector memory phenotype. Functionally, TCRs from tumors or inflamed tissues demonstrated over 10,000-fold significantly avidity compared to TCRs from healthy donors. These TCRs also exhibited higher specificity, recognizing only the cognate peptide. Finally, tumor-derived TCRs demonstrated improved yeast display and pMHC binding.Conclusions Together, these findings highlight tumor-derived TCRs as optimal candidates for the development of TCR-based T cell engagers, combining high affinity, specificity, and favorable biophysical properties.